The present invention relates to nucleic acid sequences of the aarC gene, amino acid sequences of the AarC polypeptide and to methods for screening compositions for their antimicrobial activity and for the detection of organisms susceptible to antimicrobials.
Resistance of nosocomial and community-acquired pathogens to antimicrobial agents is a serous problem with significant clinical and economic consequences. Many species are resistant to commonly used antimicrobials, and in many cases resistance to multiple classes of drugs is reported. In the past few years, a handful of organisms resistant to all known antimicrobial agents has emerged [see, Tenover et al., Am. J. Med. Sci. 311:9-16 (1996)]. Though such organisms are rare, the existence of conditions favoring the development and spread of these organisms forecasts the continued emergence of multi-drug resistance. This problem is further exacerbated by the scarcity of new classes of antimicrobial agents since many pharmaceutical manufacturers have abandoned the discovery of antimicrobial drugs in favor of identifying antifungal and antiviral drugs [see, Tenover et al., JAMA 275(4):300-4, 1996].
Antimicrobial drug resistance has been documented in both gram-negative and gram-positive bacterial pathogens. Among the clinically significant gram-negative bacteria, which account for 60% of infections treated in hospitals, resistance to multiple drugs has been reported in Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Haemophilus influenzae, and Neisseriae gonorrhoeae. Multiple-drug resistance is also found in gram-positive bacteria, such as Staphylococcus aureus, Staphylococcus hemolyticus, and Streptococcus pneumoniae which are isolated from hospital environments. Because resistance to multiple classes of drugs is rapidly spreading among clinically significant bacterial isolates, the clinical and economic consequences of multiple-drug resistance are severe.
A. Antimicrobial Drug Resistance
Three key factors have contributed to the emergence and spread of microbes which are resistant to multiple antimicrobial compounds, including mutations in common resistance genes, exchange of genetic information among microorganisms, and the increased selective pressures in institutional settings and communities.
Mutations in common resistance genes have extended the bacterial spectrum of drug resistance. Resistance genes mostly encode proteins that either inactivate antimicrobial agents or block their site of action. For example, the organism may alter the receptors to which the antimicrobial binds (e.g., conformational changes in penicillin binding proteins (PBPs), such that penicillin won""t bind), or it alter the cell membrane such that membrane transport systems are ineffective in transporting the antimicrobial across the cell membrane (e.g., resistance to tetracyclines due to the fact that the drug cannot enter the cell). Alternatively, the organism can develop enzymes which destroy or inactivate the antimicrobial (e.g., xcex2-lactamases which destroy penicillin). In addition, the organism can also alter an enzyme""s specificity for its substrate (e.g., sulfonamide-resistant bacteria often have enzymes with a high affinity for para-aminobenzoic acid (PABA), but a low affinity for sulfonamide), or altogether forego its requirement for a particular substrate (e.g., exogenous folic acid may be taken in by sulfonamide-resistant bacteria, thereby by-passing the need to take in PABA as a precursor of folic acid synthesis). Importantly, microorganisms that are resistant to a given drug may also be resistant to other drugs that share certain mechanisms of action. This cross-resistance is usually observed with drugs that are closely related chemically or that have a similar mode of binding or action.
Small changes in resistance determinants (e.g., enzymes) can also have major effects on an organism""s resistance profile to drugs which belong to different chemical classes. This is exemplified by the changes in xcex2-lactamases. These enzymes inactivate xcex2-lactam drugs such as penicillin, ampicillin, and cephalothin. Mutant forms of the xcex2-lactamases, which are referred to as extended-spectrum xcex2-lactamases (ESBLs) and which are capable of inactivating the chemically unrelated extended-spectrum cephalosporins and monobactams were reported as early as 1982. Only three amino acid differences, which reflect point mutations in the coding sequence of the xcex2-lactamase, exist between the ESBL and wild-type xcex2-lactamases.
The problem of multiple-resistant bacteria is compounded by the exchange of genetic information between bacteria. Bacteria exchange information by transformation (i.e., the uptake of naked DNA), transduction (i.e., transfer of DNA by bacteriophage), and conjugation (i.e., cell-to-cell contact). The exchange of extrachromosomal elements such as plasmids and transposons during conjugation is the most common method of resistance transfer. Although conjugation was previously thought to be limited to gram-negative bacilli, a similar transfer process has been extensively documented for gram-positive organisms whereby plasmids or independent transposable elements, often carrying multiple-resistance genes, move from one organism to another. The transfer process extends even between gram-negative and gram-positive organisms. For example, Campylobacter coli and enterococci have been shown to exchange aminoglycoside resistance genes {Trieu-Cuot et al., EMBO J. 4:3583-3587 (1985)]. Thus, a susceptible strain can acquire resistance from another resistant species or genus.
Environmental pressures encourage the emergence or acquisition of new mutations. Such pressures include the extended and prophylactic use of antimicrobials in communities, hospitals, nursing homes, day care centers and animal feedlots. In addition, many antimicrobials are bacteriostatic rather than bacteriocidal. Organisms exposed to bacteriostatic drugs remain viable, although their growth is inhibited. Because they remain viable, these organisms are provided with the opportunity to develop mechanisms of resistance to the drug. For example, the use in hospitals of antimicrobials prone to select altered resistance traits results in hospitalized patients, who are usually immunocompromised, quickly becoming colonized with resistant strains.
B. Addressing Antimicrobial Drug Resistance
Attempts to minimize the impact of multiple-drug resistance have focused on barrier isolation, improvement of antimicrobial use, and proper design and use of instruments. Barrier isolation precautions aim to contain infection by reducing a hospitalized patient""s physical contact with bacteria. Although such precautions are effective against bacteria from exogenous sources, they are of limited use in containing organisms endogenous to the patient. Furthermore, regardless of the quality of isolation precautions, they are ineffective in containing antimicrobial drug resistance which arises by mutation, genetic transfer, emergence, and selection of resistant strains. In addition, though manageable in a hospital setting, isolation precautions are often impractical in the community.
Avoiding the misuse of antimicrobials is also important in dealing with multiresistant organisms. While controlling the use of antimicrobials in hospitals may go a long way towards minimizing the problem, such control is difficult to enforce. Moreover, the benefits of controlled antimicrobial use in hospitals are often thwarted by the slower conformance in the community to judicious antimicrobial drug use.
An additional attack on problems of multiresistance includes proper design and use of instruments. Some organisms have attributes, independent of their ability to resist antimicrobials, which allow them to survive in or around instruments, thus making instruments a vehicle for dissemination of resistance and a reservoir of hospital organisms. Though better instrument design and use may reduce the dissemination of such organisms, it nevertheless does not address the development of antimicrobial drug resistance via mutation and selection.
What is needed is a new gene or gene product which can be targeted by classes of antimicrobials that are different from those currently used and to which microbial resistance is established. Discovery of such new genes and their products is particularly useful where they are present in more than one microbial strain, and found in both gram-negative and gram-positive microbes.
In one embodiment, the present invention provides a substantially purified polypeptide comprising at least a portion of the amino acid sequence of SEQ ID NO:5. In one preferred embodiment, the purified polypeptide comprises a portion of the SEQ ID NO:5 having a length greater than about 65 amino acid residues, more preferably greater than about 75 amino acid residues, and most preferably greater than about 90 amino acid residues.
In another embodiment, the invention provides an isolated polynucleotide sequence encoding a polypeptide comprising at least a portion of the amino acid sequence of SEQ ID NO:5. In one embodiment, the polynucleotide sequence is contained on a recombinant expression vector. In a further preferred embodiment, the expression vector containing the polynucleotide sequence is contained within a host cell.
In yet another embodiment, the invention provides a polynucleotide sequence that hybridizes under stringent conditions to the nucleic acid sequence of SEQ ID NO:4.
In a further embodiment, the invention provides a method of screening a compound, the method comprising: a) providing, in any order: i) bacteria containing a recombinant expression vector, wherein the vector comprises at least a portion of the oligonucleotide sequence of SEQ ID NO:4 or variants or homologs thereof; and ii) a compound suspected of having antimicrobial activity; b) contacting the bacteria with the compound; and c) detecting antimicrobial activity of the compound. In one preferred embodiment, the antimicrobial activity is bacteriostatic. In a further preferred embodiment, the antimicrobial activity is bactericidal. In yet a further preferred embodiment, the bacteria are gram negative. In a further preferred embodiment, the gram negative bacteria is Escherichia coli. In an alternative preferred embodiment, the bacteria are gram positive. In a further preferred embodiment, the gram positive bacteria is Bacillus subtilis. 
In one embodiment of the method of screening a compound, the vector further comprises a fusion sequence, wherein the fusion sequence comprises a reporter sequence operably linked to aac(2xe2x80x2)-la. In a particularly preferred embodiment, the reporter sequence is a xcex2-galactosidase sequence.
Yet another embodiment of the invention provides a method for detecting the presence of polynucleotide sequences encoding at least a portion of aarC gene in a sample, the method comprising the steps of: a) providing in any order: i) at least a portion of the nucleotide of SEQ ID NO:4, or a variant or homolog thereof; and ii) a sample suspected of containing nucleic acid corresponding to at least a portion of the polynucleotide sequence of SEQ ID NO:4 or variants or homologs thereof, b) combining the nucleotide and the sample under conditions such that a hybridization complex is formed between the nucleotide sequence and the polynucleotide; and c) detecting the hybridization complex. In one preferred embodiment, the nucleotide sequence is RNA. In another preferred embodiment, the nucleotide sequence is DNA. In one embodiment, the detected hybridization complex correlates with expression of the at least portion of the polynucleotide of SEQ ID NO:4 or variants of homologs thereof in the sample.
In another embodiment, the invention provides a purified antibody which binds specifically to a polypeptide comprising at least a portion of the amino acid sequence of SEQ ID NO:5.
In yet another embodiment, the invention provides a method for detecting the expression of AarC in a sample, the method comprising the steps of: a) providing in any order: i) an antibody which binds specifically to a polypeptide comprising at least a portion of the amino acid sequence of SEQ ID NO:5; and ii) a sample suspected of expressing AarC; b) combining the sample and the antibody under conditions such that an antibody:protein complex is formed; and c) detecting the complex wherein the presence of the complex correlates with the expression of the protein in the sample. In one preferred embodiment, the antibody is polyclonal. In another preferred embodiment, the antibody is monoclonal.
To facilitate understanding of the invention, a number of terms are defined below.
xe2x80x9cNucleic acid sequencexe2x80x9d and xe2x80x9cnucleotide sequencexe2x80x9d as used herein refer to an oligonucleotide or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin which may be single- or double-stranded, and represent the sense or antisense strand.
xe2x80x9cAmino acid sequencexe2x80x9d and xe2x80x9cpolypeptide sequencexe2x80x9d are used interchangeably herein to refer to a sequence of amino acids.
As used herein, xe2x80x9cAarCxe2x80x9d or xe2x80x9cAarC polypeptidexe2x80x9d or xe2x80x9cAarC proteinxe2x80x9d are used interchangeably to refer to the amino acid sequence of substantially purified AarC obtained from any species, particularly bacterial species which include gram negative, gram positive, aerobic, and anaerobic bacteria, and obtained from any source whether natural, synthetic, semi-synthetic or recombinant.
A xe2x80x9cvariantxe2x80x9d of AarC is defined as an amino acid sequence which differs by one or more amino acids from the AarC polypeptide sequence of SEQ ID NO:5. The variant may have xe2x80x9cconservativexe2x80x9d changes, wherein a substituted amino acid has similar structural or chemical properties, e.g., replacement of leucine with isoleucine. More rarely, a variant may have xe2x80x9cnonconservativexe2x80x9d changes, e.g., replacement of a glycine with a tryptophan. Similar minor variations may also include amino acid deletions or insertions (i.e., additions), or both. Guidance in determining which and how many amino acid residues may be substituted, inserted or deleted without abolishing biological or immunological activity may be found using computer programs well known in the art, for example, DNAStar software.
A xe2x80x9cvariantxe2x80x9d of aarC is defined as a nucleotide sequence which differs from SEQ ID NO:4, e.g., by having deletions, insertions, and substitutions that may be detected using hybridization assays. Included within this definition is the detection of alterations to the genomic DNA sequence which encodes AarC [e.g., by alterations in the pattern of restriction enzyme fragments capable of hybridizing to SEQ ID NO:4 (RFLP analysis), the inability of a selected fragment of SEQ ID NO:4 to hybridize under high stringency conditions to a sample of genomic DNA (e.g., using allele-specific oligonucleotide probes), and improper or unexpected hybridization, such as hybridization to a locus other than the normal chromosomal locus for the aarC gene (e.g., using fluorescent in situ hybridization (FISH)).
A xe2x80x9cdeletionxe2x80x9d is defined as a change in either nucleotide or amino acid sequence in which one or more nucleotides or amino acid residues, respectively, are absent.
An xe2x80x9cinsertionxe2x80x9d or xe2x80x9cadditionxe2x80x9d is that change in a nucleotide or amino acid sequence which has resulted in the addition of one or more nucleotides or amino acid residues, respectively, as compared to, for example, the naturally occurring Providencia stuartii AarC.
A xe2x80x9csubstitutionxe2x80x9d results from the replacement of one or more nucleotides or amino acids by different nucleotides or amino acids, respectively.
The term xe2x80x9cderivativexe2x80x9d of aarC as used herein refers to the chemical modification of a nucleic acid encoding AarC. Illustrative of such modifications would be replacement of hydrogen by an alkyl, acyl, or amino group. A nucleic acid derivative would encode a polypeptide which retains essential biological characteristics of natural human AarC.
As used herein the term xe2x80x9cportionxe2x80x9d when in reference to a protein (as in xe2x80x9ca portion of a given proteinxe2x80x9d) refers to fragments of that protein. The fragments may range in size from four amino acid residues to the entire amino acid sequence minus one amino acid. Thus, a protein xe2x80x9ccomprising at least a portion of the amino acid sequence of SEQ ID NO:5xe2x80x9d encompasses the full-length human AarC protein and fragments thereof.
The term xe2x80x9cportionxe2x80x9d when used in reference to a nucleotide sequence refers to fragments of that nucleotide sequence. The fragments may range in size from 5 nucleotide residues to the entire nucleotide sequence minus one nucleic acid residue.
The term xe2x80x9cbiologically activexe2x80x9d refers to a AarC molecule having structural, regulatory or biochemical functions of a naturally occurring AarC. AarC biological activity is determined, for example, by restoration of wild-type growth in cells lacking AarC activity (i.e., AarC null cells). Cells lacking AarC activity may be produced using methods well known in the art (e.g., point mutation and frame-shift mutation). Complementation is achieved by transfecting cells which lack AarC activity with an expression vector which expresses AarC, a derivative thereof, or a portion thereof. Details concerning complementation of AarC null cells is provided in Example 3 herein.
The term xe2x80x9cimmunologically activexe2x80x9d defines the capability of the natural, recombinant or synthetic AarC, or any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
The term xe2x80x9cantigenic determinantxe2x80x9d as used herein refers to that portion of a molecule that is recognized by a particular antibody (i.e., an epitope). When a protein or fragment of a protein is used to immunize a host animal, numerous regions of the protein may induce the production of antibodies which bind specifically to a given region or three-dimensional structure on the protein; these regions or structures are referred to as antigenic determinants. An antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.
The terms xe2x80x9cimmunogen,xe2x80x9d xe2x80x9cantigen,xe2x80x9d xe2x80x9cimmunogenicxe2x80x9d and xe2x80x9cantigenicxe2x80x9d refer to any substance capable of generating antibodies when introduced into an animal. By definition, an immunogen must contain at least one epitope (the specific biochemical unit capable of causing an immune response), and generally contains many more. Proteins are most frequently used as immunogens, but lipid and nucleic acid moieties complexed with proteins may also act as immunogens. The latter complexes are often useful when smaller molecules with few epitopes do not stimulate a satisfactory immune response by themselves.
The term xe2x80x9cantibodyxe2x80x9d refers to immunoglobulin evoked in animals by an immunogen (antigen). It is desired that the antibody demonstrates specificity to epitopes contained in the immunogen. The term xe2x80x9cpolyclonal antibodyxe2x80x9d refers to immunoglobulin produced from more than a single clone of plasma cells; in contrast xe2x80x9cmonoclonal antibodyxe2x80x9d refers to immunoglobulin produced from a single clone of plasma cells.
The terms xe2x80x9cspecific bindingxe2x80x9d or specifically bindingxe2x80x9d when used in reference to the interaction of an antibody and a protein or peptide means that the interaction is dependent upon the presence of a particular structure (i.e., the antigenic determinant or epitope) on the protein; in other words the antibody is recognizing and binding to a specific protein structure rather than to proteins in general. For example, if an antibody is specific for epitope xe2x80x9cAxe2x80x9d, the presence of a protein containing epitope A (or free, unlabelled A) in a reaction containing labelled xe2x80x9cAxe2x80x9d and the antibody will reduce the amount of labelled A bound to the antibody.
The term xe2x80x9crecombinant DNA moleculexe2x80x9d as used herein refers to a DNA molecule which is comprised of segments of DNA joined together by means of molecular biological techniques.
The term xe2x80x9crecombinant proteinxe2x80x9d or xe2x80x9crecombinant polypeptidexe2x80x9d as used herein refers to a protein molecule which is expressed using a recombinant DNA molecule.
As used herein, the terms xe2x80x9cvectorxe2x80x9d and xe2x80x9cvehiclexe2x80x9d are used interchangeably in reference to nucleic acid molecules that transfer DNA segment(s) from one cell to another.
The term xe2x80x9cexpression vectorxe2x80x9d or xe2x80x9cexpression cassettexe2x80x9d as used herein refers to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism. Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), and a ribosome binding site, often along with other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.
The terms xe2x80x9cin operable combinationxe2x80x9d, xe2x80x9cin operable orderxe2x80x9d and xe2x80x9coperably linkedxe2x80x9d as used herein refer to the linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced. The term also refers to the linkage of amino acid sequences in such a manner so that a functional protein is produced.
The term xe2x80x9ctransfectionxe2x80x9d as used herein refers to the introduction of foreign DNA into cells. Transfection may be accomplished by a variety of means known to the art including calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, biolistics (i.e., particle bombardment) and the like.
As used herein, the terms xe2x80x9ccomplementaryxe2x80x9d or xe2x80x9ccomplementarityxe2x80x9d are used in reference to xe2x80x9cpolynucleotidesxe2x80x9d and xe2x80x9coligonucleotidesxe2x80x9d (which are interchangeable terms that refer to a sequence of nucleotides) related by the base-pairing rules. For example, the sequence xe2x80x9cC-A-G-T,xe2x80x9d is complementary to the sequence xe2x80x9cG-T-C-A.xe2x80x9d Complementarity can be xe2x80x9cpartialxe2x80x9d or xe2x80x9ctotal.xe2x80x9d xe2x80x9cPartialxe2x80x9d complementarity is where one or more nucleic acid bases is not matched according to the base pairing rules. xe2x80x9cTotalxe2x80x9d or xe2x80x9ccompletexe2x80x9d complementarity between nucleic acids is where each and every nucleic acid base is matched with another base under the base pairing rules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as detection methods which depend upon binding between nucleic acids.
The terms xe2x80x9chomologyxe2x80x9d and xe2x80x9chomologousxe2x80x9d as used herein in reference to nucleotide sequences refer to a degree of complementarity with other nucleotide sequences. There may be partial homology or complete homology (i.e., identity). A nucleotide sequence which is partially complementary, i.e., xe2x80x9csubstantially homologous,xe2x80x9d to a nucleic acid sequence is one that at least partially inhibits a completely complementary sequence from hybridizing to a target nucleic acid sequence. The inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or Northern blot, solution hybridization and the like) under conditions of low stringency. A substantially homologous sequence or probe will compete for and inhibit the binding (i.e., the hybridization) of a completely homologous sequence to a target sequence under conditions of low stringency. This is not to say that conditions of low stringency are such that non-specific binding is permitted; low stringency conditions require that the binding of two sequences to one another be a specific (i.e., selective) interaction. The absence of non-specific binding may be tested by the use of a second target sequence which lacks even a partial degree of complementarity (e.g., less than about 30% identity); in the absence of non-specific binding the probe will not hybridize to the second non-complementary target.
An oligonucleotide sequence which is a xe2x80x9chomologxe2x80x9d of the P. Stuartii aarC gene of SEQ ID NO:4 is defined herein as an oligonucleotide sequence which exhibits greater than or equal to 50% identity to the sequence of SEQ ID NO:4 when sequences having a length of 100 bp or larger are compared. Alternatively, a homolog of SEQ ID NO:4 is defined as an oligonucleotide sequence which encodes a biologically active AarC amino acid sequence.
Low stringency conditions comprise conditions equivalent to binding or hybridization at 42xc2x0 C. in a solution consisting of 5xc3x97SSPE (43.8 g/l NaCl, 6.9 g/l NaH2PO4.H2O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS, 5xc3x97 Denhardt""s reagent [50xc3x97 Denhardt""s contains per 500 ml: 5 g Ficoll (Type 400, Pharmacia), 5 g BSA (Fraction V; Sigma)] and 100 xcexcg/ml denatured salmon sperm DNA followed by washing in a solution comprising 5xc3x97SSPE, 0.1% SDS at 42xc2x0 C. when a probe of about 500 nucleotides in length is employed.
The art knows well that numerous equivalent conditions may be employed to comprise low stringency conditions; factors such as the length and nature (DNA, RNA, base composition) of the probe and nature of the target (DNA, RNA, base composition, present in solution or immobilized, etc.) and the concentration of the salts and other components (e.g., the presence or absence of formamide, dextran sulfate, polyethylene glycol), as well as components of the hybridization solution may be varied to generate conditions of low stringency hybridization different from, but equivalent to, the above listed conditions. In addition, the art knows conditions which promote hybridization under conditions of high stringency (e.g., increasing the temperature of the hybridization and/or wash steps, the use of formamide in the hybridization solution, etc.).
When used in reference to a double-stranded nucleic acid sequence such as a cDNA or genomic clone, the term xe2x80x9csubstantially homologousxe2x80x9d refers to any probe which can hybridize to either or both strands of the double-stranded nucleic acid sequence under conditions of low stringency as described above.
When used in reference to a single-stranded nucleic acid sequence, the term xe2x80x9csubstantially homologousxe2x80x9d refers to any probe which can hybridize (ie., it is the complement of) the single-stranded nucleic acid sequence under conditions of low stringency as described above.
As used herein, the term xe2x80x9chybridizationxe2x80x9d is used in reference to the pairing of complementary nucleic acids using any process by which a strand of nucleic acid joins with a complementary strand through base pairing to form a hybridization complex. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is impacted by such factors as the degree of complementarity between the nucleic acids, stringency of the conditions involved, the Tm of the formed hybrid, and the G:C ratio within the nucleic acids.
As used herein the term xe2x80x9chybridization complexxe2x80x9d refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bounds between complementary G and C bases and between complementary A and T bases; these hydrogen bonds may be further stabilized by base stacking interactions. The two complementary nucleic acid sequences hydrogen bond in an antiparallel configuration. A hybridization complex may be formed in solution (e.g., C0t or R0t analysis) or between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized to a solid support [e.g., a nylon membrane or a nitrocellulose filter as employed in Southern and Northern blotting, dot blotting or a glass slide as employed in in situ hybridization, including FISH (fluorescent in situ hybridization)].
As used herein, the term xe2x80x9cTmxe2x80x9d is used in reference to the xe2x80x9cmelting temperature.xe2x80x9d The melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands. The equation for calculating the Tm of nucleic acids is well known in the art. As indicated by standard references, a simple estimate of the Tm value may be calculated by the equation: Tm=81.5+0.41(% G+C), when a nucleic acid is in aqueous solution at 1 M NaCl [see e.g., Anderson and Young, Quantitative Filter Hybridization, in Nucleic Acid Hybridization (1985)]. Other references include more sophisticated computations which take structural as well as sequence characteristics into account for the calculation of Tm.
As used herein the term xe2x80x9cstringencyxe2x80x9d is used in reference to the conditions of temperature, ionic strength, and the presence of other compounds such as organic solvents, under which nucleic acid hybridizations are conducted. xe2x80x9cStringencyxe2x80x9d typically occurs in a range from about Tm to about 20xc2x0 C. to 25xc2x0 C. below Tm. As will be understood by those of skill in the art, a stringent hybridization can be used to identify or detect identical polynucleotide sequences or to identify or detect similar or related polynucleotide sequences. The stringent conditions are chosen such that SEQ ID NO:4 or fragments thereof will hybridize to sequences encoding AarC but not to sequences encoding GpcE (i.e., SEQ ID NO:6) or RNA equivalents of GpcE. When fragments of SEQ ID NO:4 are employed in hybridization reactions, the stringent conditions include the choice of fragments of SEQ ID NO:4 to be used. Fragments of SEQ ID NO:4 which contain unique sequences (i.e., regions which are either non-homologous to or which contain less than about 50% homology or complementarity with SEQ ID NOs:4) are preferentially employed. SEQ ID NO: 4 represents a DNA sequence encoding the AarC protein; this DNA sequence can be found in GenBank under accession number U67933. Fragments of SEQ ID NO:4 which contain unique sequences (i.e., regions which are either non-homologous to or which contain less than 50% homology or complementarity with SEQ ID NO:4) are preferentially employed. Conditions of xe2x80x9cweakxe2x80x9d or xe2x80x9clowxe2x80x9d stringency are often required with nucleic acids that are derived from organisms that are genetically diverse, as the frequency of complementary sequences is usually low between such organisms.
As used herein, the term xe2x80x9camplifiable nucleic acidxe2x80x9d is used in reference to nucleic acids which may be amplified by any amplification method. It is contemplated that xe2x80x9camplifiable nucleic acidxe2x80x9d will usually comprise xe2x80x9csample template.xe2x80x9d
As used herein, the term xe2x80x9csample templatexe2x80x9d refers to nucleic acid originating from a sample which is analyzed for the presence of a target sequence of interst. In contrast, xe2x80x9cbackground templatexe2x80x9d is used in reference to nucleic acid other than sample template which may or may not be present in a sample. Background template is most often inadvertent. It may be the result of carryover, or it may be due to the presence of nucleic acid contaminants sought to be purified away from the sample. For example, nucleic acids from organisms other than those to be detected may be present as background in a test sample.
xe2x80x9cAmplificationxe2x80x9d is defined as the production of additional copies of a nucleic acid sequence and is generally carried out using polymerase chain reaction technologies well known in the art [Dieffenbach CW and GS Dveksler (1995) PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview N.Y.]. As used herein, the term xe2x80x9cpolymerase chain reactionxe2x80x9d (xe2x80x9cPCRxe2x80x9d) refers to the method of K. B. Mullis U.S. Pat. Nos. 4,683,195 and 4,683,202, hereby incorporated by reference, which describe a method for increasing the concentration of a segment of a target sequence in a mixture of genomic DNA without cloning or purification. The length of the amplified segment of the desired target sequence is determined by the relative positions of two oligonucleotide primers with respect to each other, and therefore, this length is a controllable parameter. By virtue of the repeating aspect of the process, the method is referred to as the xe2x80x9cpolymerase chain reactionxe2x80x9d (hereinafter xe2x80x9cPCRxe2x80x9d). Because the desired amplified segments of the target sequence become the predominant sequences (in terms of concentration) in the mixture, they are said to be xe2x80x9cPCR amplifiedxe2x80x9d.
With PCR, it is possible to amplify a single copy of a specific target sequence in genomic DNA to a level detectable by several different methodologies (e.g., hybridization with a labeled probe; incorporation of biotinylated primers followed by avidin-enzyme conjugate detection; incorporation of 32P-labeled deoxynucleotide triphosphates, such as dCTP or dATP, into the amplified segment). In addition to genomic DNA, any oligonucleotide sequence can be amplified with the appropriate set of primer molecules. In particular, the amplified segments created by the PCR process itself are, themselves, efficient templates for subsequent PCR amplifications.
As used herein, the term xe2x80x9cprimerxe2x80x9d refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, (i.e., in the presence of nucleotides and an inducing agent such as DNA polymerase and at a suitable temperature and pH). The primer is preferably single stranded for maximum efficiency in amplification, but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent. The exact lengths of the primers will depend on many factors, including temperature, source of primer and the use of the method.
As used herein, the term xe2x80x9cprobexe2x80x9d refers to an oligonucleotide (i.e., a sequence of nucleotides), whether occurring naturally as in a purified restriction digest or produced synthetically, recombinantly or by PCR amplification, which is capable of hybridizing to another oligonucleotide of interest. A probe may be single-stranded or double-stranded. Probes are useful in the detection, identification and isolation of particular gene sequences. It is contemplated that any probe used in the present invention will be labelled with any xe2x80x9creporter molecule,xe2x80x9d so that is detectable in any detection system, including, but not limited to enzyme (e.g., ELISA, as well as enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems. It is not intended that the present invention be limited to any particular detection system or label.
As used herein, the terms xe2x80x9crestriction endonucleasesxe2x80x9d and xe2x80x9crestriction enzymesxe2x80x9d refer to bacterial enzymes, each of which cut double-stranded DNA at or near a specific nucleotide sequence.
DNA molecules are said to have xe2x80x9c5xe2x80x2 endsxe2x80x9d and xe2x80x9c3xe2x80x2 endsxe2x80x9d because mononucleotides are reacted to make oligonucleotides in a manner such that the 5xe2x80x2 phosphate of one mononucleotide pentose ring is attached to the 3xe2x80x2 oxygen of its neighbor in one direction via a phosphodiester linkage. Therefore, an end of an oligonucleotide is referred to as the xe2x80x9c5xe2x80x2 endxe2x80x9d if its 5xe2x80x2 phosphate is not linked to the 3xe2x80x2 oxygen of a mononucleotide pentose ring. An end of an oligonucleotide is referred to as the xe2x80x9c3xe2x80x2 endxe2x80x9d if its 3xe2x80x2 oxygen is not linked to a 5xe2x80x2 phosphate of another mononucleotide pentose ring. As used herein, a nucleic acid sequence, even if internal to a larger oligonucleotide, also may be said to have 5xe2x80x2 and 3xe2x80x2 ends. In either a linear or circular DNA molecule, discrete elements are referred to as being xe2x80x9cupstreamxe2x80x9d or 5xe2x80x2 of the xe2x80x9cdownstreamxe2x80x9d or 3xe2x80x2 elements. This terminology reflects the fact that transcription proceeds in a 5xe2x80x2 to 3xe2x80x2 fashion along the DNA strand. The promoter and enhancer elements which direct transcription of a linked gene are generally located 5xe2x80x2 or upstream of the coding region. However, enhancer elements can exert their effect even when located 3xe2x80x2 of the promoter element and the coding region. Transcription termination and polyadenylation signals are located 3xe2x80x2 or downstream of the coding region.
As used herein, the term xe2x80x9can oligonucleotide having a nucleotide sequence encoding a genexe2x80x9d means a nucleic acid sequence comprising the coding region of a gene, i.e. the nucleic acid sequence which encodes a gene product. The coding region may be present in either a cDNA, genomic DNA or RNA form. When present in a DNA form, the oligonucleotide may be single-stranded (i.e., the sense strand) or double-stranded. Suitable control elements such as enhancers/promoters, splice junctions, polyadenylation signals, etc. may be placed in close proximity to the coding region of the gene if needed to permit proper initiation of transcription and/or correct processing of the primary RNA transcript. Alternatively, the coding region utilized in the expression vectors of the present invention may contain endogenous enhancers/promoters, splice junctions, intervening sequences, polyadenylation signals, etc. or a combination of both endogenous and exogenous control elements.
As used herein, the term xe2x80x9cregulatory elementxe2x80x9d refers to a genetic element which controls some aspect of the expression of nucleic acid sequences. For example, a promoter is a regulatory element which facilitates the initiation of transcription of an operably linked coding region. Other regulatory elements are splicing signals, polyadenylation signals, termination signals, etc.
Transcriptional control signals in eukaryotes comprise xe2x80x9cpromoterxe2x80x9d and xe2x80x9cenhancerxe2x80x9d elements. Promoters and enhancers consist of short arrays of DNA sequences that interact specifically with cellular proteins involved in transcription [Maniatis, T. et al., Science 236:1237 (1987)]. Promoter and enhancer elements have been isolated from a variety of eukaryotic sources including genes in plant, yeast, insect and mammalian cells and viruses (analogous control elements, i.e., promoters, are also found in prokaryotes). The selection of a particular promoter and enhancer depends on what cell type is to be used to express the protein of interest.
The presence of xe2x80x9csplicing signalsxe2x80x9d on an expression vector often results in higher levels of expression of the recombinant transcript. Splicing signals mediate the removal of introns from the primary RNA transcript and consist of a splice donor and acceptor site [Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York (1989) pp. 16.7-16.8]. A commonly used splice donor and acceptor site is the splice junction from the 16S RNA of SV40.
Efficient expression of recombinant DNA sequences in eukaryotic cells requires expression of signals directing the efficient termination and polyadenylation of the resulting transcript. Transcription termination signals are generally found downstream of the polyadenylation signal and are a few hundred nucleotides in length. The term xe2x80x9cpoly A sitexe2x80x9d or xe2x80x9cpoly A sequencexe2x80x9d as used herein denotes a DNA sequence which directs both the termination and polyadenylation of the nascent RNA transcript. Efficient polyadenylation of the recombinant transcript is desirable as transcripts lacking a poly A tail are unstable and are rapidly degraded. The poly A signal utilized in an expression vector may be xe2x80x9cheterologousxe2x80x9d or xe2x80x9cendogenous.xe2x80x9d An endogenous poly A signal is one that is found naturally at the 3xe2x80x2 end of the coding region of a given gene in the genome. A heterologous poly A signal is one which is isolated from one gene and placed 3xe2x80x2 of another gene.
The term xe2x80x9ctransfectionxe2x80x9d or xe2x80x9ctransfectedxe2x80x9d refers to the introduction of foreign DNA into a cell.
As used herein, the terms xe2x80x9cnucleic acid molecule encoding,xe2x80x9d xe2x80x9cDNA sequence encoding,xe2x80x9d and xe2x80x9cDNA encodingxe2x80x9d refer to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide (protein) chain. The DNA sequence thus codes for the amino acid sequence.
As used herein, the term xe2x80x9cantisensexe2x80x9d is used in reference to RNA sequences which are complementary to a specific RNA sequence (e.g., MRNA). Antisense RNA may be produced by any method, including synthesis by splicing the gene(s) of interest in a reverse orientation to a viral promoter which permits the synthesis of a coding strand. Once introduced into a cell, this transcribed strand combines with natural mRNA produced by the cell to form duplexes. These duplexes then block either the further transcription of the mRNA or its translation. In this manner, mutant phenotypes may be generated. The term xe2x80x9cantisense strandxe2x80x9d is used in reference to a nucleic acid strand that is complementary to the xe2x80x9csensexe2x80x9d strand. The designation (xe2x88x92) (i.e., xe2x80x9cnegativexe2x80x9d) is sometimes used in reference to the antisense strand, with the designation (+) sometimes used in reference to the sense (i.e., xe2x80x9cpositivexe2x80x9d) strand.
The term xe2x80x9cSouthern blotxe2x80x9d refers to the analysis of DNA on agarose or acrylamide gels to fractionate the DNA according to size, followed by transfer and immobilization of the DNA from the gel to a solid support, such as nitrocellulose or a nylon membrane. The immobilized DNA is then probed with a labeled oligodeoxyribonucleotide probe or DNA probe to detect DNA species complementary to the probe used. The DNA may be cleaved with restriction enzymes prior to electrophoresis. Following electrophoresis, the DNA may be partially depurinated and denatured prior to or during transfer to the solid support. Southern blots are a standard tool of molecular biologists [J. Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N.Y., pp 9.31-9.58].
The term xe2x80x9cNorthern blotxe2x80x9d as used herein refers to the analysis of RNA by electrophoresis of RNA on agarose gels to fractionate the RNA according to size followed by transfer of the RNA from the gel to a solid support, such as nitrocellulose or a nylon membrane. The immobilized RNA is then probed with a labeled oligodeoxyribonucleotide probe or DNA probe to detect RNA species complementary to the probe used. Northern blots are a standard tool of molecular biologists [J. Sambrook, J. et al. (1989) supra, pp 7.39-7.52].
The term xe2x80x9creverse Northern blotxe2x80x9d as used herein refers to the analysis of DNA by electrophoresis of DNA on agarose gels to fractionate the DNA on the basis of size followed by transfer of the fractionated DNA from the gel to a solid support, such as nitrocellulose or a nylon membrane. The immobilized DNA is then probed with a labeled oligo-ribonuclotide probe or RNA probe to detect DNA species complementary to the ribo probe used.
The term xe2x80x9cisolatedxe2x80x9d when used in relation to a nucleic acid, as in xe2x80x9can isolated oligonucleotidexe2x80x9d refers to a nucleic acid sequence that is identified and separated from at least one contaminant nucleic acid with which it is ordinarily associated in its natural source. Isolated nucleic acid is nucleic acid present in a form or setting that is different from that in which it is found in nature. In contrast, non-isolated nucleic acids are nucleic acids such as DNA and RNA which are found in the state they exist in nature. For example, a given DNA sequence (e.g., a gene) is found on the host cell chromosome in proximity to neighboring genes; RNA sequences, such as a specific mRNA sequence encoding a specific protein, are found in the cell as a mixture with numerous other mRNAs which encode a multitude of proteins. However, isolated nucleic acid encoding a AarC polypeptide includes, by way of example, such nucleic acid in cells ordinarily expressing a AarC polypeptide where the nucleic acid is in a chromosomal or extrachromosomal location different from that of natural cells, or is otherwise flanked by a different nucleic acid sequence than that found in nature. The isolated nucleic acid or oligonucleotide may be present in single-stranded or double-stranded form. When an isolated nucleic acid or oligonucleotide is to be utilized to express a protein, the oligonucleotide will contain at a minimum the sense or coding strand (i.e., the oligonucleotide may be single-stranded). Alternatively, it may contain both the sense and anti-sense strands (ie., the oligonucleotide may be double-stranded).
As used herein, the term xe2x80x9cpurifiedxe2x80x9d or xe2x80x9cto purifyxe2x80x9d refers to the removal of undesired components from a sample. For example, where recombinant AarC polypeptides are expressed in bacterial host cells, the AarC polypeptides are purified by the removal of host cell proteins thereby increasing the percent of recombinant AarC polypeptides in the sample.
As used herein, the term xe2x80x9csubstantially purifiedxe2x80x9d refers to molecules, either nucleic or amino acid sequences, that are removed from their natural environment, isolated or separated, and are at least 60% free, preferably 75% free, and more preferably 90% free from other components with which they are naturally associated. An xe2x80x9cisolated polynucleotidexe2x80x9d is therefore a substantially purified polynucleotide.
As used herein the term xe2x80x9ccoding regionxe2x80x9d when used in reference to a structural gene refers to the nucleotide sequences which encode the amino acids found in the nascent polypeptide as a result of translation of a mRNA molecule. The coding region is bounded, in eukaryotes, on the 5xe2x80x2 side by the nucleotide triplet xe2x80x9cATGxe2x80x9d which encodes the initiator methionine and on the 3xe2x80x2 side by one of the three triplets which specify stop codons (i.e., TAA, TAG, TGA).
As used herein, the term xe2x80x9cstructural genexe2x80x9d refers to a DNA sequence coding for RNA or a protein. In contrast, xe2x80x9cregulatory genesxe2x80x9d are structural genes which encode products which control the expression of other genes (e.g., transcription factors).
As used herein, the term xe2x80x9cgenexe2x80x9d means the deoxyribonucleotide sequences comprising the coding region of a structural gene and including sequences located adjacent to the coding region on both the 5xe2x80x2 and 3xe2x80x2 ends for a distance of about 1 kb on either end such that the gene corresponds to the length of the full-length mRNA. The sequences which are located 5xe2x80x2 of the coding region and which are present on the mRNA are referred to as 5xe2x80x2 non-translated sequences. The sequences which are located 3xe2x80x2 or downstream of the coding region and which are present on the mRNA are referred to as 3xe2x80x2 non-translated sequences. The term xe2x80x9cgenexe2x80x9d encompasses both cDNA and genomic forms of a gene. A genomic form or clone of a gene contains the coding region interrupted with non-coding sequences termed xe2x80x9cintronsxe2x80x9d or xe2x80x9cintervening regionsxe2x80x9d or xe2x80x9cintervening sequences.xe2x80x9d Introns are segments of a gene which are transcribed into heterogenous nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or xe2x80x9cspliced outxe2x80x9d from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript. The mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.
In addition to containing introns, genomic forms of a gene may also include sequences located on both the 5xe2x80x2 and 3xe2x80x2 end of the sequences which are present on the RNA transcript. These sequences are referred to as xe2x80x9cflankingxe2x80x9d sequences or regions (these flanking sequences are located 5xe2x80x2 or 3xe2x80x2 to the non-translated sequences present on the mRNA transcript). The 5xe2x80x2 flanking region may contain regulatory sequences such as promoters and enhancers which control or influence the transcription of the gene. The 3xe2x80x2 flanking region may contain sequences which direct the termination of transcription, posttranscriptional cleavage and polyadenylation.
The term xe2x80x9csamplexe2x80x9d as used herein is used in its broadest sense and includes environmental and biological samples. Environmental samples include material from the environment such as soil and water. Biological samples may be animal, including, human, fluid (e.g., blood, plasma and serum), solid (e.g., stool), tissue, liquid foods (e.g. milk), and solid foods (e.g., vegetables). A biological sample suspected of containing nucleic acid encoding AarC may comprise a cell, tissue extract, body fluid, chromosomes or extrachromosomal elements isolated from a cell, genomic DNA (in solution or bound to a solid support such as for Southern blot analysis), RNA (in solution or bound to a solid support such as for Northern blot analysis), CDNA (in solution or bound to a solid support) and the like.
The term xe2x80x9cantimicrobialxe2x80x9d and xe2x80x9cantibacterialxe2x80x9d are used interchangeably to refer to a composition which reduces the rate of growth of an organism compared to the rate of growth of the organism in the absence of the composition. An antimicrobial can be natural (e.g., derived from bacteria), synthetic, or recombinant. An antimicrobial can be bacteriostatic, bactericidal or both. An antimicrobial is bacteriostatic if it inhibits cell division without affecting the viability of the inhibited cell. An antimicrobial is bactericidal if it causes cell death. Cell death is commonly detected by the absence of cell growth in liquid growth medium (e.g., absence of turbidity) or on a solid surface (e.g., absence of colony formation on agar). Those of skill in the art know that a composition which is bacteriostatic at a given concentration may be bactericidal at a higher concentration. Certain bacteriostatic compositions are not bactericidal at any concentration.
The term xe2x80x9cbacteriaxe2x80x9d and xe2x80x9cbacteriumxe2x80x9d refer to all prokaryotic organisms, including those within all of the phyla in the Kingdom Procaryotae. It is intended that the term encompass all microorganisms considered to be bacteria including Mycoplasma, Chlamydia, Actinomyces, Streptomyces, and Rickettsia. All forms of bacteria are included within this definition including cocci, bacilli, spirochetes, spheroplasts, protoplasts, etc. Also included within this term are prokaryotic organisms which are gram negative or gram positive. xe2x80x9cGram negativexe2x80x9d and xe2x80x9cgram positivexe2x80x9d refer to staining patterns with the Gram-staining process which is well known in the art [Finegold and Martin, Diagnostic Microbiology, 6th Ed. (1982), C V Mosby St. Louis, pp 13-15]. xe2x80x9cGram positive bacteriaxe2x80x9d are bacteria which retain the primary dye used in the Gram stain, causing the stained cells to appear dark blue to purple under the microscope. xe2x80x9cGram negative bacteriaxe2x80x9d do not retain the primary dye used in the Gram stain, but are stained by the counterstain. Thus, gram negative bacteria appear red.
The present invention is premised on the fortuitous discovery of the aarC gene in Providencia stuartii. Data presented herein demonstrates that the wild-type form of the aarC gene shown in FIG. 2 (SEQ ID NO:4) is essential to the viability of Providencia stuartii. A transversion in the nucleic acid sequence of this gene leading to a single amino acid substitution resulted in a very slow growing phenotype. In addition, disruption of the aarC gene by insertion of a nucleic acid sequence within the aarC gene resulted in cell death.
Providencia stuartii is a member of the family Enterobacteriaceae. It is a nosocomial pathogen which exhibits multiple resistance to antibiotics and is responsible for a variety of human infections, particularly nosocomial urinary tract infections which are more prevalent in older patients. P. stuartii has also been reported in hospital patients with bacteraemia, septicaemia associated with burns, and in non-specific diarrhea in infants under one year of age. Despite the apparently low virulence of the organism, it can cause extensive and long-term outbreaks of infection with fatalities in specialized hospital units such as intensive therapy units and bums units [Hawkey, P. Journal of Antimicrob. Chemotherapy 13:209-226 (1984)].
P. stuartii is resistant to several classes of antimicrobials, including the aminoglycoside and cephalosporin antibiotics [Penner et al. Antimicrobial Agents and Chemotherapy 22:218-221 (1982)] and substituted penicillin [Hawkey et al., Antimicrobial Agents and Chemotherapy 23:619-621 (1983)]. For example, many of the first- and second-generation cephalosporins are susceptible to the xcex2-lactamases produced by P. stuartii, with 97% resistance rates being reported to cephalothin, 82% to cephaloridine, 94% to cefazolin, and 100% to cephalexin [Hawkey, P. Journal of Antimicrob. Chemotherapy 13:209-226 (1984)]. In addition, and in common with other gram-negative bacteria, P. stuartii is resistant to aminoglycoside antibiotics. High proportions of P. stuartii (80-100%) were resistant to gentamicin both in wards where the drug is widely used and in others (e.g., geriatric wards) in which it was not used at all.
The resistance of P. stuartii to aminoglycoside antimicrobials results partly from the production of the chromosomally encoded aminoglycoside-inactivating enzyme aminoglycoside N-acetyltransferase (2xe2x80x2), i.e., AAC(2xe2x80x2)-la, which is encoded by the aac(2xe2x80x2)-la gene (SEQ ID NO:12) (FIG. 8) previously described by Rather et al. (1993) J. Bacteriol. 175:6492-6498. This enzyme was initially identified by its role in aminoglycoside resistance [Chevereau, M. et al., Biochemistry 13:598-603 (1974), Rather, P. et al., J. Bacteriol. 175:6492-6498 (1993), Yamaguchi, M. et al., J. Antibiotics 27:507-515 (1974)]. AAC(2xe2x80x2)-la is presumed to acetylate aminoglycosides because of their structural similarity with the peptidoglycan substrates of this enzyme. This enzyme is believed to have, in addition to its known role in aminoglycoside resistance, an important housekeeping function in the O-acetylation of peptidoglycan.
The aac(2xe2x80x2)-la gene is universally present in the chromosome of P. stuartii regardless of resistance phenotype. Regulation of aac(2xe2x80x2)-la expression is complex. Recessive mutations in at least four loci (i.e., aarA, aarB, aarD, and aarG) have been identified that increase aac(2xe2x80x2)-la mRNA accumulation [Macinga et al. (1981) Mol. Microbiol. 19:511-520; Rather et al. (1993) J. Bacteriol. 175:6492-6498]. In addition, a gene (aarP) encoding a transcriptional activator (AarP) has also been identified [Macinga, D. R. et al. J. Bacteriol. 177:3407-3413 (1995)]. An additional level of regulation of the aac(2xe2x80x2)-la gene includes repression mediated by a diffusible extracellular factor, AR-factor, which acts by an unknown mechanism to decrease aac(2xe2x80x2)-la mRNA accumulation as cells approach high density.
The present invention provides nucleic acid sequences encoding P. stuartii AarC and its variants. The aarC gene was discovered in the course of an investigation of negative regulators of the aac(2xe2x80x2)-la gene. The present invention provides results which demonstrate that the wild-type aarC gene is essential for cell viability. A missense allele (aarC1) resulted in an 8.9-fold increase in xcex2-galactosidase accumulation from an aac(2xe2x80x2)-lacZ transcriptional fusion. Northern blot analysis demonstrated that this increase was specific to aac(2xe2x80x2)-lacZ mRNA accumulation. mRNA encoding AarP (a transcription activator of the aac(2xe2x80x2)-la gene) was also elevated in P. stuartii cells containing the aarC1 allele. Both the elevation of aac(2xe2x80x2)-lacZ mRNA and aarP mRNA were observed only in cells at high density. While not intending to limit the invention to any particular mechanism, the observation that aarC-mediated regulation of aac(2xe2x80x2)-la and of aarP is specific to cells at high cell density suggests that aarC may act in a pathway by which P. stuartii responds to extracellular factor(s) involved in regulating aac(2xe2x80x2)-la expression.
The wild-type aarC gene (SEQ ID NO:4) of the present invention was isolated by complementation and was shown to encode a predicted AarC polypeptide sequence (SEQ ID NO:5) of 366 amino acids with a molecular weight of 39815 Da. The predicted AarC polypeptide sequence was homologous to amino acid sequences from gram negative and gram positive bacteria. Thus, the AarC polypeptide exhibited 88% amino acid homology to the previously identified GcpE protein of Escherichia coli, a gene product whose function is unknown and reported to be essential [Baker, J. et al., FEMS Microbiolgy Letters 92:175-180 (1992), Eisenbeis, S. J. et al., Mol. Gen. Genet. 183:115-122 (1981)], and 86% homology to a gene product from Haemophilus influenzae. AarC also showed 51% homology to a B. subtilis protein.
As demonstrated herein, the E. coli gcpE gene was able to functionally complement the aarC1 allele in P. stuartii. The aarC1 allele was identified as a T to G transversion that resulted in a valine to glycine substitution at position 136 in the AarC polypeptide. The present invention demonstrates that the aarC gene is essential for cell viability as construction of a disrupted copy (aarC::lacZ) of the gene was possible only in cells that carried an episomal copy of aarC or gcpE. The essential nature of aarC in P. stuartii and the conservation of this gene product in three different gram negative organisms as well as in a gram positive organism make the aarC gene and its product, the AarC polypeptide, attractive targets for the development of new antimicrobials.
I. The aarC Nucleotide Sequence
The nucleic acid sequence of the aarC gene (SEQ ID NO:4) and the amino acid sequence of the AarC polypeptide (SEQ ID NO:5) encoded by this gene are shown in FIG. 2. While the precise function of the AarC polypeptide is unclear, data presented in this invention demonstrates that it performs a function which is essential to the microbe""s survival. This conclusion is based in part on (a) the homology of the AarC polypeptide to proteins from the gram negative E. coli and H. influenzae and the gram positive B. subtilis, (b) the very slow growing cell phenotype which resulted from a single point mutation in the aarC sequence, (c) cell death as a result of disruption of the aarC gene, and (d) generation of viable cells by complementation of a disrupted aarC gene with an episomal copy of the wild-type aarC gene.
The present invention contemplates any nucleic acid sequence which encodes the AarC polypeptide sequence or its variants; these nucleic acid sequences are used to make recombinant molecules which express the AarC polypeptide. For example, one of ordinary skill in the art would recognize that the redundancy of the genetic code permits an enormous number of nucleic acid sequences which encode the AarC polypeptide. Thus, codons which are different from those shown in FIG. 2 may be used to increase the rate of expression of the nucleotide sequence in a particular prokaryotic or eukaryotic expression host which has a preference for particular codons. Additionally, alternative codons may also be used in eukaryotic expression hosts to generate splice variants of recombinant RNA transcripts which have more desirable properties (e.g., longer or shorter half-life) than transcripts generated using the sequence depicted in FIG. 2. In addition, different codons may also be desirable for the purpose of altering restriction enzyme sites or, in eukaryotic expression hosts, of altering glycosylation patterns in translated polypeptides.
Variants of the nucleotide sequence of FIG. 2 are also included within the scope of this invention. These variants include, but are not limited to, nucleotide sequences having deletions, insertions or substitutions of different nucleotides or nucleotide analogs as long as the biological activity of the translation product of the nucleotide sequence is maintained.
This invention is not limited to the aarC sequence (SEQ ID NO:4) but specifically includes nucleic acid homologs which are capable of hybridizing to the nucleotide sequence of FIG. 2, and to portions, variants and derivatives thereof. Those skilled in the art know that different hybridization stringencies may be desirable. For example, whereas higher stringencies may be preferred to reduce or eliminate non-specific binding between the nucleotide sequence of FIG. 2 and other nucleic acid sequences, lower stringencies may be preferred to detect a larger number of nucleic acid sequences having different homologies to the nucleotide sequence of FIG. 2.
Fragments of the aarC sequence (SEQ ID NO:4) are also specifically contemplated to be within the scope of this invention. It is preferred that the fragments have a length equal to or greater than 10 nucleotides and show greater than 50% homology to SEQ ID NO:5. These fragments are exemplified by, but not restricted to, the sequence 5xe2x80x2-CACTGTGCGG-3xe2x80x2 (SEQ ID NO:11) which is located between the nucleotide 303-312 of SEQ ID NO:2.
The present invention further contemplates antisense molecules comprising the nucleic acid sequence complementary to at least a portion of the polynucleotide of SEQ ID NO:4.
The scope of this invention further encompasses nucleotide sequences containing the nucleotide sequence of FIG. 2, portions, variants, derivatives and homologs thereof, ligated to one or more heterologous sequences as part of a fusion gene. Such fusion genes may be desirable, for example, to detect expression of sequences which form part of the fusion gene. Examples of a heterologous sequence include the reporter sequence encoding the enzyme xcex2-galactosidase or the enzyme luciferase. Fusion genes may also be desirable to facilitate purification of the expressed protein. For example, the heterologous sequence of protein A allows purification of the fusion protein on immobilized immunoglobulin. Other affinity traps are well known in the art and can be utilized to advantage in purifying the expressed fusion protein. For example, pGEX vectors (Promega, Madison Wis.) may be used to express the AarC polypeptides as a fusion protein with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. Proteins made in such systems are designed to include heparin, thrombin or factor XA protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.
The nucleotide sequence shown in FIG. 2, portions, variants, derivatives and homologs thereof can be synthesized by synthetic chemistry techniques which are commercially available and well known in the art. The nucleotide sequence of synthesized sequences may be confirmed using commercially available kits as well as from methods well known in the art which utilize enzymes such as the Klenow fragment of DNA polymerase I, Sequenase(copyright), Taq DNA polymerase, or thermostable T7 polymerase. Capillary electrophoresis may also be used to analyze the size and confirm the nucleotide sequence of the products of nucleic acid synthesis. Synthesized sequences may also be amplified using the polymerase chain reaction (PCR) as described by Mullis [U.S. Pat. No. 4,683,195] and Mullis et al. [U.S. Pat. No. 4,683,202], the ligase chain reaction [LCR; sometimes referred to as xe2x80x9cLigase Amplification Reactionxe2x80x9d (LAR)] described by Barany, Proc. Natl. Acad. Sci., 88:189 (1991); Barany, PCR Methods and Applic., 1:5 (1991); and Wu and Wallace, Genomics 4:560 (1989).
It is readily appreciated by those in the art that the aarC nucleotide sequences of the present invention may be used in a variety of ways. For example, fragments of the sequence of at least about 10 bp, more usually at least about 15 bp, and up to and including the entire (i.e., full-length) sequence can be used as probes for the detection and isolation of complementary genomic DNA sequences from P. stuartii as well as other bacteria. Genomic sequences are isolated by screening a genomic library containing bacterial DNA with all or a portion of the aarC sequence (SEQ ID NO:4). In addition to screening genomic libraries, the aarC sequence can also be used to screen cDNA libraries made using bacterial RNA.
The aarC sequence is also useful in directing the synthesis of AarC. The AarC polypeptide finds use in producing AarC antibodies for diagnostic purposes such as detecting infections with bacteria which express AarC. The aarC sequence is also useful for the screening of antimicrobials and the detection of microbes which contain aarC sequences and their homologs. These uses are described in the following sections.
II. The AarC Polypeptide Sequence
The present invention provides the polypeptide sequence (SEQ ID NO:5) of AarC as shown in FIG. 2 and specifically contemplates variants thereof. For example, AarC variants included within the scope of this invention include AarC polypeptide sequences containing deletions, insertion or substitutions of amino acid residues which result in a polypeptide that is functionally equivalent to the AarC polypeptide of FIG. 2. For example, amino acids may be substituted for other amino acids having similar characteristics of polarity, charge, solubility, hydrophobicity, hydrophilicity and/or amphipathic nature. Alternatively, substitution of amino acids with other amino acids having one or more different characteristic may be desirable for the purpose of producing a polypeptide which is secreted from the cell in order to, for example, simplify purification of the polypeptide.
The AarC polypeptide sequence of FIG. 2 and its functional variants may be made using chemical synthesis. For example, peptide synthesis of the AarC polypeptide, in whole or in part, can be performed using solid-phase techniques well known in the art. Synthesized polypeptides can be substantially purified by high performance liquid chromatography (HPLC) techniques, and the composition of the purified polypeptide confirmed by amino acid sequencing. One of skill in the art would recognize that variants of the AarC polypeptide can be produced by manipulating the polypeptide sequence during and/or after its synthesis.
AarC and its functional variants can also be produced by an expression system. Expression of AarC may be accomplished by inserting the nucleotide sequence of FIG. 2, its variants, portions, derivatives or homologs into appropriate vectors to create expression vectors, and transfecting the expression vectors into host cells.
Expression vectors can be constructed using techniques well known in the art [Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview N.Y.; Ausubel et al. (1989) Current Protocols in Molecular Biology, John Wiley and Sons, New York N.Y.]. Briefly, the nucleic acid sequence of interest is placed in operable combination with transcription and translation regulatory sequences. Regulatory sequences include initiation signals such as start (i.e., ATG) and stop codons, promoters which may be constitutive (i.e., continuously active) or inducible, as well as enhancers to increase the efficiency of expression, and transcription termination signals. Transcription termination signals must be provided downstream from the structural gene if the termination signals of the structural gene are not included in the expression vector. Expression vectors may become integrated into the genome of the host cell into which they are introduced, or are present as unintegrated vectors. Typically, unintegrated vectors are transiently expressed and regulated for several hours (eg., 72 hours) after transfection.
The choice of promoter is governed by the type of host cell to be transfected with the expression vector. Host cells include bacterial, yeast, plant, insect, and mammalian cells. Transfected cells may be identified by any of a number of marker genes. These include antibiotic (e.g., gentamicin, penicillin, and kanamycin) resistance genes as well as marker or reporter genes (e.g., xcex2-galactosidase and luciferase) which catalyze the synthesis of a visible reaction product.
Expression of the gene of interest by transfected cells may be detected either indirectly using reporter genes, or directly by detecting mRNA or protein encoded by the gene of interest. Indirect detection of expression may be achieved by placing a reporter gene in tandem with the sequence encoding AarC under the control of a single promoter. Expression of the reporter gene indicates expression of the tandem AarC sequence. It is preferred that the reporter gene have a visible reaction product. For example, cells expressing the reporter gene xcex2-galactosidase produce a blue color when grown in the presence of X-Gal, whereas cells grown in medium containing luciferin will fluoresce when expressing the reporter gene luciferase.
Direct detection of AarC expression can be achieved using methods well known to those skilled in the art. For example, mRNA isolated from transfected cells can be hybridized to labelled oligonucleotide probes and the hybridization detected. Alternatively, polyclonal or monoclonal antibodies specific for AarC can be used to detect expression of the AarC polypeptide using enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS).
Recombinant AarC and its variants which are expressed by the host cell can be purified either from the culture medium, if the expression construct directs its secretion into culture medium, or from the host cell using purification techniques known in the art. For example, AarC polypeptide may be expressed as a fusion protein with heterologous metal chelating peptides (ie., polyhistidine tracts) or with protein A domains, and purified on commercially available immobilized metals or immunoglobulins, respectively.
Those skilled in the art recognize that the AarC polypeptide sequences of the present invention are useful in generating antibodies which find use in detecting bacteria that express AarC or proteins homologous thereto. Such detection is particularly useful in the choice of antimicrobials to be used in a clinical context. For example, a clinical sample (e.g., a urethral or genital exudate, blood, wound culture, or respiratory culture) which shows the presence of proteins reactive with AarC antibodies indicates the presence of bacteria which contain aarC. This further suggests that it may be advantageous to administer antimicrobials which alter AarC activity to the patient from whom the sample was derived.
III. Methods For Screening Compounds For Antimicrobial Activity
This invention contemplates methods for screening compounds for antimicrobial activity which alter the activity of AarC. As used herein, the term xe2x80x9cantimicrobialxe2x80x9d is intended to mean a compound which is bacteriostatic, bactericidal or both. Such compounds include natural compounds isolated from viral, bacterial, fungal, plant and animal sources, as well as synthetic compounds. Neither the source, chemical structure, or mechanism of action of the antimicrobial is critical to this invention. For example, an antimicrobial may alter AarC activity by modifying transcriptional, translational or post-translational mechanisms either singly or in combination.
Several methods are provided herein for using the sequences of the present invention to screen potential antimicrobials which alter AarC biological activity. One method contemplates using bacterial cells which contain chromosomal aarC are transformed with an expression vector which contains both aarC and aac(2xe2x80x2)-la nucleotide sequences in either a xe2x80x9cdilution testxe2x80x9d or a xe2x80x9cdisc diffusion test.xe2x80x9d Bacterial cells containing such an expression vector can be generated as described above. In the dilution test, it is preferred that three types of bacterial cell are used. The first cell type contains chromosomal aarC. The second and third cell types contain, in addition to the chromosomal aarC, a single copy or multiple copies of the expression vector respectively. Each of the three cell types is separately contacted with the antimicrobial by inoculating a suspension of each cell type into a series of tubes containing a range of known concentrations of the potential antimicrobial to be tested.
After incubation, antimicrobial activity is detected by determination of the inhibition of visible growth as measured by lack of turbidity. If the compound has no effect at any concentration tested on the growth of any of the three cell types, then the compound is not antimicrobial.
Alternatively, if at a given concentration the potential antimicrobial inhibits the growth of each of the three cell types, then the compound""s activity could either specifically target AarC activity, or be independent of AarC. To ascertain whether the potential antimicrobial targets AarC activity, differential growth of bacterial cells which contain a single copy and multiple copies of the expression vector is examined in a series of concentrations (lower than the concentration which resulted in growth inhibition of each of the three cell types) of the potential antimicrobial. If the growth of cells containing a single copy and multiple copies of the vector is equally inhibited at each of the tested concentrations, the potential antimicrobial probably does not target AarC. On the other hand, if growth inhibition by the potential antimicrobial of cells containing a single copy of the vector is greater than the growth inhibition observed in cells containing multiple copes of the vector, the antimicrobial likely targets AarC. This is because the additional AarC polypeptide which is produced by cells containing multicopy vectors would alleviate the inhibition of AarC expressed by a single copy of the vector.
Similarly, if at a given concentration of the potential antimicrobial, growth inhibition is greatest in cells which do not contain the vector, with cells containing a single copy of the vector showing intermediate inhibition and with cells containing multiple vector copies showing the least inhibition, then the antimicrobial likely targets AarC. This is because the increased activity of AarC, which is concomitant with increased plasmid copy number, in cells containing multiple expression vectors alleviates the tested compound""s antimicrobial activity
In the disc diffusion test, a paper disk containing a specified amount (not concentration) of the antimicrobial to be tested is applied to an agar surface that has been freshly inoculated with bacterial cells which contain either a single copy or multiple copies of an expression vector which expresses both aarC and aac(2xe2x80x2)-la nucleotide sequences. The antimicrobial is allowed to diffuse into the medium over an 18- to 24-hour period resulting in a zone of inhibition at the point at which a critical concentration of the antimicrobial inhibits bacterial growth. If a zone of inhibition is observed on plates containing either cells which have a single copy or multiple copies of the expression vector, the antimicrobial probably does not affect AarC activity. On the other hand, if a zone of growth inhibition is observed only on plates in which the bacteria contain a single copy of the expression vector, and not on plates in which the bacteria contain multiple copies of the vector, then the antimicrobial likely targets AarC activity.
Yet other methods are contemplated for screening antimicrobials which alter AarC activity. One embodiment contemplates using bacterial cells which have been transformed with expression vectors which contain aarC and a fusion gene of aac(2xe2x80x2)-la and a reporter gene. For example, preliminary screening of antimicrobials can be performed by using bacterial cells which contain an expression vector that expresses aac(2xe2x80x2)-lacZ and a single copy of an expression vector which expresses wild type aarC. These cells are grown on an agar surface containing X-Gal and treated either with different amounts of the antimicrobial to be tested or with buffer alone. The formation of blue colonies on plates treated with the antimicrobial, and of white colonies on control plates treated with buffer alone suggests that the antimicrobial alters AarC activity. To confirm that antimicrobial activity targets AarC activity, a secondary screening step is performed. In that step the effect of the antimicrobial is compared using bacterial cells containing an expression vector which expresses aac(2xe2x80x2)-lacZ as well as either a single copy or multiple copies of an expression vector which expresses wild type aarC cells. Following parallel treatment of the two cell types with different concentrations of the antimicrobial, and growth on agar containing X-Gal, the color of the rings around the colonies arising from each of the cells is visually compared. If a given concentration of the antimicrobial results in the development of blue rings on plates of cells containing a single copy of the aarC expression vector, and in rings having a relatively reduced blue intensity on plates of cells containing multiple copies of the aarC expression vector, then the test antimicrobial activity likely interferes with AarC activity. This is because the higher concentration of AarC produced by cells containing a multiple copy of an aarC expression vector relative to the AarC concentration produced by cells expressing a single copy of the aarC expression vector would be expected to alleviate the antimicrobial""s effect on AarC.
Having screened a potential antimicrobial compound for its effect on AarC, the bacteriostatic and bactericidal activity of the antimicrobial compound can be further examined using techniques well known in the art [Snyder et al., In Modern Pharmacology, 2d Ed., C. R. Craig and R. E. Stitzel, (eds.), Little, Brown and Company, Boston, pp. 631-640 (1986); Conte et al., Manual of Antibiotics and Infectious Diseases, 6th Ed., Lea and Febier, Philadelphia, pp. 135-152 (1988)]. To determine the compound""s bacteriostatic activity in relation to a particular bacterium, a cell suspension of the particular bacterium is introduced into a number of tubes which contain a range of known concentrations of the antimicrobial, usually as an integral power of 2 (e.g., 128 xcexcg/ml) and decreasing on a log2 basis (i.e., 64, 32, 16, 8, 4, etc.) to the lowest concentration to be tested. After incubation, the lowest concentration inhibiting visible growth by turbidity is referred to as the minimum inhibitory concentration (MIC). Inhibition of visible bacterial growth indicates bacteriostatic activity.
To assess bactericidal activity, an aliquot is taken from a tube showing bacteriostatic activity as described above, and this aliquot is added to agar plates. If growth occurs, then the agent is bacteriostatic. However, if no growth occurs, the agent is bactericidal. The minimal bactericidal concentration (MBC), which is also referred to as the minimum lethal concentration (MLC), is defined by the lowest concentration of antimicrobial yielding less than or equal to 0.1% survivor organisms from the original inoculum of approximately 100,00 organisms.
The above-described methods of determining bacteriostatic and bactericidal activities have been reviewed [Woods, Infect. Dis. Clin. North. Am. 9(3):463-481 (1995)] and standardized for antimicrobial testing of aerobic and facultatively anaerobic bacteria [National Committee for Clinical Laboratory Standards: Performance Standards For Antimicrobial Disk Susceptibility Tests: Approved Standard, 5th ed. NCCLS Document M2-A5. Villanova, Pa., NCCLS (1993); National Committee for Clinical Laboratory Standards. Methods For Dilution Antimicrobial Susceptibility Tests For Bacteria That Grow Aerobically: Approved Standard, 3nd ed. NCCLS Document M7-A3. Villanova, Pa., NCCLS, (1993)], anaerobic bacteria [National Committee for Clinical Laboratory Standards: Methods For Antimicrobial Susceptibility Testing Of Anaerobic Bacteria: Approved Standard, 3nd ed. NCCLS Document M11-A3. Villanova, Pa., NCCLS (1993)] and for mycobacteria [National Committee for Clinical Laboratory Standards: Antimicobacterial Susceptibility Testing: Proposed Standard. NCCLS Document P24-P. Villanova, Pa., NCCLS (1989)].
IV. Methods For Detecting Microbes Containing The aarC Nucleotide Sequence
The present invention provides methods for the detection of microbes which contain the aarC nucleotide sequence shown in FIG. 2, fragments, variants, derivatives and homologs thereof. Cells which contain the aarC coding sequence may be identified by a variety of procedures know to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridization as well as amplification (e.g., PCR) using DNA probes (e.g., oligonucleotide or oligomer probes or amplimers), mRNA probes and fragments of the sequence encoding AarC. These probes and fragments can be made using a wide variety of techniques known in the art such as chemical synthesis, restriction digestion and expression of the aarC sequence, or any portion of it, in an expression vector. Labelling of the synthesized or expressed probes and aarC fragments can be achieved using oligolabeling, end-labeling or PCR amplification using a labeled nucleotide.
Having generated labelled probes, microbes present in a sample (e.g., urethral exudate, blood, urine, wound culture, respiratory culture, genital specimen or feces specimen) can be tested for the presence of the aarC sequence using Southern or reverse Northern analysis of isolated plasmid or total cellular DNA, or using Northern analysis of MRNA.
Alternatively, whole-cell lysates of colonies may be used. Colonies can be either grown on filters or spotted onto filters [Moseley et al., J. Infect. Dis. 142:892-898 (1980)] either directly [Perine et al. J. Infect. Diseases 152(1):59-63 (1985)] or following overnight culture [Gootz et al., Antimicrob. Agents and Chemother. 28(1):69-73 (1985)]. Briefly, a solid support such as nitrocellulose paper or a nylon membrane is inoculated with a clinical specimen, or with a broth culture of a clinical specimen suspected of containing bacteria. The cells are lysed onto the nitrocellulose paper and the DNA denatured, for example by treatment with NaOH. The support is treated with pronase and chloroform to lower background non-specific binding of DNA probes to colony material. Prehybridization and hybridization of the filters (ie., support) with oligonucleotides, oligomers, or portions of the aarC sequence is then performed using standard techniques. Hybridization is detected using any one of many available methods, such as by imaging radioactive probes using X-ray films (i.e., autoradiography).
It will be apparent to one skilled in the art that detection of bacteria which harbor the aarC sequence or its homolog in a sample derived from a subject demonstrates that it is beneficial to administer antimicrobials which alter AarC activity to the subject. In addition, such detection also permits monitoring the subject for the presence of the bacteria during and after administration of the antimicrobial.
V. Methods For Detecting Microbes Containing The AarC Polypeptide Sequence
This invention also contemplates the detection of AarC and its variants in bacteria using AarC antibodies. These antibodies include, but are not limited to, polyclonal and monoclonal antibodies. In addition, chimeric antibodies may be produced, for example, by splicing mouse antibody genes to human antibody genes. Single chain antibodies against AarC are also contemplated as well as antibody fragments generated by pepsin digestion of the antibody molecule or by reduction of the disulfide bridges of the Fab fragments.
For the production of AarC antibodies, any antigenic portion of the AarC sequence of FIG. 2 can be used either alone (to produce an antibody against AarC) or fused with amino acids of another protein e.g., glutathione (to produce an antibody against the chimeric molecule). Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab fragments and fragments produced by a Fab expression library. Neutralizing antibodies, ie., those which inhibit dimer formation, are especially preferred for diagnostics and therapeutics.
AarC polypeptide to be used for antibody induction need not retain biological activity; however, the protein fragment, or oligopeptide must be antigenic. Peptides used to induce specific antibodies may have an amino acid sequence consisting of at least five amino acids, preferably at least 10 amino acids. Preferably, they should mimic a portion of the amino acid sequence of the natural protein and may contain the entire amino acid sequence of a small, naturally occurring molecule. Short stretches of AarC amino acids may be fused with those of another protein such as keyhole limpet hemocyanin and antibody produced against the chimeric molecule.
For the production of antibodies, various hosts including goats, rabbits, rats, mice, etc may be immunized by injection with Aarc or any portion, fragment or oligopeptide which retains immunogenic properties. Depending on the host species, various adjuvants may be used to increase immunological response. Such adjuvants include but are not limited to Freund""s, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. BCG (Bacillus Calmette-Guerin) and Corynebacterium parvum are potentially useful adjuvants.
Monoclonal antibodies to AarC may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include but are not limited to the hybridoma technique originally described by Koehler and Milstein (1975 Nature 256:495-497), the human B-cell hybridoma technique (Kosbor et al. (1983) Immunol Today 4:72; Cote et al. (1983) Proc Natl Acad Sci 80:2026-2030) and the EBV-hybridoma technique [Cole et al. (1985) Monoclonal Antibodies and Cancer Therapy, Alan R Liss Inc, New York N.Y., pp 77-96].
AarC antibodies can be used to detect AarC polypeptide in clinical samples such as body fluids or extracts of cells or tissues using several known techniques such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS). Reporter molecules known in the art can be joined to the polypeptides and antibodies to facilitate detection of polypeptide-antibody binding.